Elevated levels of soluble P-selectin in mice alter blood-brain barrier function, exacerbate stroke, and promote atherosclerosis by Janka Kisucka, Anil K. Chauhan, Bing-Qiao Zhao, Ian S. Patten, Ayce Yesilaltay, Monty Krieger, and Denisa D. Wagner Blood Volume 113(23):6015-6022 June 4, 2009 ©2009 by American Society of Hematology

Increased BBB permeability in P-selΔCT/ΔCT mice. Increased BBB permeability in P-selΔCT/ΔCT mice. (A) Quantification of EBD accumulation in the brain of P-selΔCT/ΔCT mice compared with WT. Data represent EBD extravasation calculated from A620 OD/g. n = 17 for WT and n = 31 for P-selΔCT/ΔCT mice. (B) Comparison of EBD accumulation between the right (RH, ▴) and left hemisphere (LH, ▵) in each animal. Ovals indicate the animals with a difference between RH and LH. (C) Representative images from confocal microscopy in the cortex of 4- to 5-month-old mice injected with EBD (red) 6 hours before perfusion with large dextran (green) at the time of sacrifice. Top panels show section of cerebrum from a WT; bottom panels are from P-selΔCT/ΔCT mice. Arrow points to the accumulation of EBD outside of the vessel in the left hemisphere of the P-selΔCT/ΔCT mice. Data represent mean ± SEM. Janka Kisucka et al. Blood 2009;113:6015-6022 ©2009 by American Society of Hematology

Stroke infarct volume is increased in P-selΔCT/ΔCT mice. Stroke infarct volume is increased in P-selΔCT/ΔCT mice. Intraluminal MCAO model of stroke on P-selΔCT/ΔCT mice and age-matched sibling WT controls. (A) Representative photographs of TTC staining of sections showing enhanced infarct volume in a P-selΔCT/ΔCT mouse brain, compared with a WT mouse brain. (B) White arrow indicates small spontaneous infarct present in the uninjured hemisphere of 1 P-selΔCT/ΔCT mouse. (C) Quantitative image analysis of the infarction area. n = 7 in each group. Error bars indicate SEM. Janka Kisucka et al. Blood 2009;113:6015-6022 ©2009 by American Society of Hematology

Behavior of P-selΔCT/ΔCT mice in the social recognition test is severely impaired. Behavior of P-selΔCT/ΔCT mice in the social recognition test is severely impaired. (A) Total time(s) spent by adult mice investigating a juvenile. Males and females were analyzed separately in 2 trials (I and II). Error bars indicate SEM. (B) Aggressive behavior (adult mouse attacking juvenile) is shown as the percentage of mice that showed aggressive behavior toward the juvenile. n = 7-21 males; n = 4-8 females. Janka Kisucka et al. Blood 2009;113:6015-6022 ©2009 by American Society of Hematology

Plasma lipids and lipoprotein profiles in P-sel+/+/apoE−/− and P-selΔCT/ΔCT/apoE−/− mice. Plasma lipids and lipoprotein profiles in P-sel+/+/apoE−/− and P-selΔCT/ΔCT/apoE−/− mice. Plasma was collected from individual mice of both sexes, fasted for 4 hours. (A) Total cholesterol, unesterified cholesterol, phospholipid, and triglyceride levels in plasma from P-sel+/+/apoE−/− (■) and P-selΔCT/ΔCT/apoE−/− (□) mice. Data represent mean ± SEM (n = 6-9). Error bars indicate SEM. (B) Plasma samples from P-sel+/+/apoE−/− (●) and P-selΔCT/ΔCT/apoE−/− (○) individual animals were size fractionated by FPLC, and the total cholesterol content of each fraction was determined. The chromatograms are the average of multiple individually determined profiles (n = 4). Approximate elution positions of native very low density lipoprotein (VLDL), IDL/LDL, and HDL particles are indicated by brackets. Janka Kisucka et al. Blood 2009;113:6015-6022 ©2009 by American Society of Hematology

Atherosclerosis is accelerated in P-selΔCT/ΔCT/apoE−/− mice compared with P-sel+/+/apoE−/−. Atherosclerosis is accelerated in P-selΔCT/ΔCT/apoE−/− mice compared with P-sel+/+/apoE−/−. Animals were maintained on normal chow, and their aortic sinus lesions were evaluated at 16 weeks of age. (A) Quantitative image analysis of aortic sinus lesion area. Male group showed 2.6-fold increased size of the plaque area. Female mice had 1.8-fold larger lesions than control mice. (B) Measurement of percentage of the aortic sinus circumference covered by atherosclerotic plaques. (C) Representative images of Oil Red-O staining of the aortic sinus. Representative images, showing an increase in plaque area in P-selΔCT/ΔCT/apoE−/− mice compared with P-sel+/+/apoE−/− mice (female mice). (D) Quantitative image analysis of macrophage content in the aortic sinus atherosclerotic plaques. Data represent mean ± SEM. Janka Kisucka et al. Blood 2009;113:6015-6022 ©2009 by American Society of Hematology